The form of tuberculin discovered by Robert Koch in 1890 and introduced as a skin test by Clemens von Pirquet in 1907 could not be cultured with pure results and led to a disproportionate number of false negatives. Seibert, working as an assistant to Esmond R. Long at the University of Chicago and the University of Pennsylvania, improved the quality of the purified proteins several times over a few years by use of ammonium sulfate and different filters, until in 1940 she finally generated a batch of the protein isolate which is still the international standard reference for the diagnostic substance.